Capillary electrophoresis has developed into a highly effective means of analyzing biological samples. Capillary electrophoresis encompasses a large variety of separation modes, many of which provide separations that equal or exceed the quality of separations that can be performed on slab gels. One form of capillary electrophoresis that is particularly suitable and convenient for many separations is capillary zone electrophoresis (CZE). Other forms include capillary isoelectric focusing, capillary gel electrophoresis, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electroosmotic chromatography. Capillary electrophoresis further offers an easy interface to digital computers and the consequent opportunities for automated control and sophisticated data handling, processing and display. The long separation path in a capillary permits the separation of a large number of components in a single sample, including components which are closely related. Also, separations can be performed in relatively short periods of time by using high voltages, since the small diameter and thin wall of a capillary provide efficient removal of the joule heat generated by the voltage. Capillaries are also well suited for on-line detection of the separated species by passing a light beam either through the capillary and directly into a detector or into the capillary to activate a fluorescently labelled species.
When the inner wall of the capillary carries a fixed electrical charge, electroosmosis occurs. Electroosmosis is a bulk flow of the solvent in which the analytes migrate. Electroosmosis is particularly pronounced in capillaries made of silica-containing materials, unless special efforts are made to coat the inner wall of the capillary or otherwise shield the charge on the inner wall. In untreated silica capillaries, the charge on the wall is negative, resulting in a bulk flow from the positive anode to the negative cathode. Because of this bulk flow, both positively and negatively charged analytes can be loaded at the anode and detected near the cathode. Although negatively charged analytes tend to migrate toward the anode, they are carried toward the cathode by the bulk flow if the magnitude of the velocity of the bulk flow exceeds that of the negative analyte.
Positively charged analytes, however, are not well suited to separations in capillaries with negatively charged inner walls since the particles tend to adhere to the walls. Were it not for this problem, the electroosmotic flow would increase the apparent rate at which positively charged analytes migrate from anode to cathode. Negatively charged analytes by contrast are repelled from the negatively charged inner wall, thereby significantly reducing the adherence of analytes to the wall.
The time taken for an analyte to reach the detector window is a non-linear function of both the electroosmotic and electrophoretic components of the velocity of the analyte. The sum of these two components appears in the denominator of the equation for the migration time. As the sum approaches zero, therefore (i.e., when the components become equal in magnitude but opposite in direction), the migration time increase toward infinity. When the electrophoretic component exceeds the electroosmotic component in magnitude, the analyte does not move through the capillary toward the cathode but instead returns to the inlet vial at the anode end of the capillary.
Variability in the magnitude of electroosmotic component limits the use of migration time as a means of identifying analytes. To eliminate the variability, electrophoretic velocity can be used instead of migration time. The electrophoretic or "actual" velocity is the difference between the total or "apparent" velocity and the electroosmotic velocity. (The term "velocity" is used herein to denote a directional value, i.e., one bearing a positive sign to denote one direction or negative sign to denote the opposite direction.) Under non-varying conditions, the electrophoretic velocity is an appropriate quantity for identifying analytes in a sample. When conditions vary, the variations can often be eliminated by using the ratio of the electrophoretic velocity of the analyte to the electrophoretic velocity of a standard compound run under the same conditions, preferably as part of the same sample. This ratio is referred to as a normalized velocity. An alternative to velocity is mobility, which is the ratio of velocity to field strength, and capable of normalization in the same manner as the velocity. The normalized velocity of a given analyte is equal to its normalized mobility.
Patient samples are analyzed by capillary electrophoresis in clinical laboratories either to screen for suspected conditions or to monitor the state of a known condition. For screening, the results are compared with established clinical standards, and for monitoring they may also be compared with previous results from the same patient. For this comparison, numerical values produced by mathematical manipulation of the data or visual displays produced from the data may be used. While the generation of accurate numerical values and appropriate visual displays share similar needs, the visual display model serves better to illustrate the need to express the information appropriately. In a particularly useful visual display referred to as an overlay, the electropherogram produced from a patient sample is superimposed either over a reference electropherogram or over an electropherogram generated from a sample collected at a different time from the same patient. For this overlay, it is important that the x-axis represent a quantity that is easily associated with the identity of a particular analyte and that the y-axis represent a quantity that is easily associated with the amount to the analyte present. The preferred method uses normalization of both axes and particularly normalized mobility for the x-axis.
A typical example of a method for normalization of migration time combined with zero correction is described in Chen, U.S. Pat. No. 5,139,630 (Beckman Instruments, Inc., issued Aug. 18, 1992). According to the Chen procedure, two marker species are added to the sample prior to separation. One of the marker species is uncharged and therefore has no electrophoretic mobility of its own, its travel being due solely to the bulk movement of the running buffer (i.e., the electroosmotic flow). The other marker species is charged with a charge density greater than that of the original components of the sample and thereby travels at a rate representing the sum of its electrophoretic velocity and the electroosmotic flow velocity. The detection peaks corresponding to the two markers thus bracket the peaks of the sample components, and the time axes of peak patterns for different samples are zeroed to the peak of the uncharged marker and normalized to the peak of the charged marker. These two peaks therefore occur at the same time in each electropherogram and the locations of all other peaks (and hence the identities of the solutes represented by the other peaks) are determined relative to these two.
One of the difficulties with peak normalization based on migration time is that it does not correct an inherent problem that arises when comparing capillary electrophoresis to electrophoresis performed in planar (slab) gels. In planar gels, the separation between components of the sample mixture is measured by terminating the electric current while the components are spread out along the gel, and detection and identification of the components is then performed by scanning the gel and recording the positions of the components on the gel (i.e., the migration distance of each component from the edge where the sample is first applied). In capillary electrophoresis, the electric current is maintained until all components have passed through the detection window, and detection and identification are therefore based on migration time rather than migration distance. With certain types of samples, this creates a noticeable difference in the appearance of the electropherogram. In serum protein separations, for instance, the gamma region in a capillary separation looks considerably different from the gamma region produced by a planar gel separation. The lack of familiar shapes makes capillary electrophoresis less appealing.
Another difficulty with the use of migration time as the x-axis arises from the fact that migration time depends strongly on the surface charge density on the inner wall of the capillary and that charge density can vary considerably from capillary to capillary, from run to run using the same capillary, and from sample to sample within the same run. Electrophoretic mobility, by contrast, does not depend on the surface charge density of the capillary wall. Although migration time normalization reduces the consequences of this dependency significantly, it does not eliminate the consequences entirely.
Difficulties arise with the use of a neutral marker for zero correction. The addition of an uncharged species to the sample to serve as a neutral marker often interferes with the separation pattern sought to be detected by producing a peak that overlaps or otherwise obscures or distorts sample component peaks occurring close by, such as the gamma region in a five-band serum protein separation. Native artifacts of the electropherogram such as a small peak near the beginning of the electropherogram can be used instead of an added uncharged species. These artifacts may also interfere with the pattern, and special adjustments such as increasing the detection wavelength are often done to eliminate them.